Dataset Information


Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.

ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.


PROVIDER: S-EPMC6520371 | BioStudies | 2019-01-01

REPOSITORIES: biostudies

Similar Datasets

1000-01-01 | S-EPMC4860831 | BioStudies
2019-01-01 | S-EPMC6419801 | BioStudies
2017-01-01 | S-EPMC5569088 | BioStudies
2018-01-01 | S-EPMC6108506 | BioStudies
2015-01-01 | S-EPMC4417674 | BioStudies
2017-01-01 | S-EPMC5215926 | BioStudies
1000-01-01 | S-EPMC5611662 | BioStudies
1000-01-01 | S-EPMC5385939 | BioStudies
1000-01-01 | S-EPMC5155318 | BioStudies
2019-01-01 | S-EPMC6344613 | BioStudies